Substrate targeting mechanism of a DesCEND pathway

摘要 这项对授权R01GM135592的补充申请提出了获得一种液体色层 蛋白质纯化系统支持重点研究底物靶向的研究目标 干细胞因子泛素连接酶F-box亚单位FBXO31在一种新的下行途径中的作用机制。通过 我们在系统水平和分子水平上鉴定和表征了FBXO31的底物 建议阐明FBXO31-degron对的互补结构决定因素 底物专一性决定蛋白质的识别和命运。为了实现我们的目标，我们必须拥有 能够从细胞裂解物和细胞裂解物的复杂起始材料中获得高纯度的蛋白质样本 重组蛋白表达系统。这项提议要求提供资金来替换一个14岁的孩子(最近 已停用)Akta FPLC蛋白质纯化仪器，其类似但最新的版本来自同一 制造商。拟议的系统，Akta Pure，是一种多功能和强大的高效液相色谱系统。 用于蛋白质和蛋白质复合体的可靠纯化。它涵盖了广泛的层析技术 并满足提供最高纯度所需的自动化要求。这台仪器将 确保继续进行父级奖项中提出的生化、生物物理和结构研究。

Abstract This request for a supplement to the grant R01GM135592 proposes the acquisition of a liquid chromatography protein purification system to support the research aims that focus on investigating the substrate targeting mechanisms of the F-box subunit FBXO31 of the SCF ubiquitin ligase in a novel DesCEND pathway. By identifying and characterizing substrates of FBXO31 at both a systems level and the molecular level, we proposed to elucidate the complementary structural determinants of the FBXO31–degron pair that confer substrate specificity and dictate protein recognition and fate. To achieve our goals, it is vital that we have the ability to obtain highly purified protein samples from complex starting materials derived from cell lysates and recombinant protein expression systems. This proposal requests funds to replace a 14-year old (and recently defunct) AKTA FPLC protein purification instrument with a similar but up-to-date version from the same manufacturer. The proposed system, the AKTA pure, is a versatile and powerful liquid chromatography system for the reliable purification of proteins and protein complexes. It covers a broad range of chromatography applications and meets the automation requirements needed to deliver the highest purity. The instrument will ensure the continued biochemical, biophysical and structural studies proposed in the parent award.